A third signal that can be provided by IL-12 or type I IFN is required for differentiation of naive CD8 T cells responding to Ag and costimulation. The cytokines program development of function and memory within 3 days of initial stimulation, and we show here that programming involves regulation of a common set of ∼355 genes including T-bet and eomesodermin. Much of the gene regulation program is initiated in response to Ag and costimulation within 24 h but is then extinguished unless a cytokine signal is available. Histone deacetylase inhibitors mimic the effects of IL-12 or type I IFN signaling, indicating that the cytokines relieve repression and allow continued gene expression by promoting increased histone acetylation. In support of this, increased association of acetylated histones with the promoter loci of granzyme B and eomesodermin is shown to occur in response to IL-12, IFN-α, or histone deacetylase inhibitors. Thus, IL-12 and IFN-α/β enforce in common a complex gene regulation program that involves, at least in part, chromatin remodeling to allow sustained expression of a large number of genes critical for CD8 T cell function and memory.

We used microarray technology to measure mRNA decay rates in resting and activated T lymphocytes in order to better understand the role of mRNA decay in regulating gene expression. Purified human T lymphocytes were stimulated for 3 h with medium alone, with an anti-CD3 antibody, or with a combination of anti-CD3 and anti-CD28 antibodies. Actinomycin D was added to arrest transcription, and total cellular RNA was collected at discrete time points over a 2 h period. RNA from each point was analyzed using Affymetrix oligonucleotide arrays and a first order decay model was used to determine the half-lives of approximately 6000 expressed transcripts. We identified hundreds of short-lived transcripts encoding important regulatory proteins including cytokines, cell surface receptors, signal transduction regulators, transcription factors, cell cycle regulators and regulators of apoptosis. Approximately 100 of these short-lived transcripts contained ARE-like sequences. We also identified numerous transcripts that exhibited stimulus-dependent changes in mRNA decay. In particular, we identified hundreds of transcripts whose steady-state levels were repressed following T cell activation and were either unstable in the resting state or destabilized following cellular activation. Thus, rapid mRNA degradation appears to be an important mechanism for turning gene expression off in an activation-dependent manner.

Following infection with most reovirus strains, viral protein synthesis is robust, even when cellular translation is inhibited. To gain further insight into pathways that regulate translation in reovirus-infected cells, we performed a comparative microarray analysis of cellular gene expression following infection with two strains of reovirus that inhibit host translation (clone 8 and clone 87) and one strain that does not (Dearing). Infection with clone 8 and clone 87 significantly increased the expression of cellular genes characteristic of stress responses, including the integrated stress response. Infection with these same strains decreased transcript and protein levels of P58 IPK , the cellular inhibitor of the eukaryotic initiation factor 2␣ (eIF2␣) kinases PKR and PERK. Since infection with host shutoff-inducing strains of reovirus impacted cellular pathways that control eIF2␣ phosphorylation and unphosphorylated eIF2␣ is required for translation initiation, we examined reovirus replication in a variety of cell lines with mutations that impact eIF2␣ phosphorylation. Our results revealed that reovirus replication is more efficient in the presence of eIF2␣ kinases and phosphorylatable eIF2␣. When eIF2␣ is phosphorylated, it promotes the synthesis of ATF4, a transcription factor that controls cellular recovery from stress. We found that the presence of this transcription factor increased reovirus yields 10-to 100-fold. eIF2␣ phosphorylation also led to the formation of stress granules in reovirus-infected cells. Based on these results, we hypothesize that eIF2␣ phosphorylation facilitates reovirus replication in two ways-first, by inducing ATF4 synthesis, and second, by creating an environment that places abundant reovirus transcripts at a competitive advantage for limited translational components.As representative members of the Reoviridae family, mammalian orthoreoviruses (reoviruses) have genomes composed of 10 segments of double-stranded RNA (dsRNA) that are surrounded by two concentric protein capsids (reviewed in reference 54). In natural infections, reoviruses replicate in cells of the respiratory or enteric tract; however, in experimental infections of neonatal mice, their tropism is much broader. Pathogenesis studies with the mouse model have demonstrated that reoviruses replicate in cells of the brain, heart, liver, muscle, and pancreas (reviewed in reference 73). At the cellular level, the consequences of reovirus infection have been extensively analyzed and include the inhibition of DNA synthesis, cell cycle arrest at the G 2 /M stage, apoptosis induction, translational inhibition, and type

AU-rich elements found in the 3-untranslated regions of cytokine and proto-oncogene transcripts regulate mRNA degradation and function as binding sites for the mRNA-stabilizing protein HuA and the mRNA-destabilizing protein tristetraprolin. Experiments were performed to evaluate the expression of HuA and tristetraprolin in purified human T lymphocytes and to evaluate the ability of these proteins to recognize specific AU-rich sequences. HuA is a predominantly nuclear protein that can also be found in the cytoplasm of resting T lymphocytes. Within 1 h after stimulation of T lymphocytes with anti-T cell receptor antibodies or a combination of a phorbol myristate acetate and ionomycin, an increase in cytoplasmic HuA RNA-binding activity was observed. Although absent in resting cells, cytoplasmic tristetraprolin protein was detected 3-6 h following activation. HuA recognized specific AU-rich sequences found in c-jun or c-myc mRNA that were poorly recognized by tristetraprolin. In contrast, tristetraprolin recognized an AU-rich sequence in interleukin-2 mRNA that was poorly recognized by HuA. Both HuA and tristetraprolin, however, recognized AUrich sequences from c-fos, interleukin-3, tumor necrosis factor-␣, and granulocyte/macrophage colony-stimulating factor mRNA. HuA may transiently stabilize a subset of AU-rich element-containing transcripts following T lymphocyte activation, and tristetraprolin may subsequently mediate their degradation.